Method and apparatus of a defined serumfree medical solution

ABSTRACT

A defined serumfree medical solution for applications in Ophthalmology, that contains one or more cell nutrient supplements which maintains and enhances the preservation of eye tissues, including human corneal tissues at low temperatures (2° C. to 15° C.). This solution is composed of a defined aqueous nutrient and electrolyte solution, supplemented with a glycosaminoglycan(s), a deturgescent agent(s), an energy source(s), a buffer system(s), an antioxidant(s), membrane stabilizing components, antibiotic(s), ATP precursors and nutrient cell supplements.

This application is a continuation of U. S. Ser. No. 07/487,919, filedMar. 5, 1990, now U.S. Pat. No. 5,104,787 entitled "Method and Apparatusfor a Defined Serumfree Medical Solution Useful for CornealPreservation", to the same Applicants.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the preservation of eye tissue in adefined nutritive. aqueous medical solution, and more particularly,relates to the preservation and enhancement of human corneal tissue,specified as the time between removal from the donor andtransplantation.

2. Description of the Prior Art

Keratoplasty, or transplantation of the cornea, has been effective inproviding visual rehabilitation to many who suffer from cornealdisorders. This procedure has gained widespread acceptance but has beenseverely hampered by the universally inconsistent availability of donortissue. This problem made the development of a storage solutionimperative. The development of MK™-preservation medium, and subsequentchondroitin sulfate-containing media, has positively impacted theavailability of quality donor tissue. Much research in this area hasbeen undertaken with a view towards prolonging donor storage time andyet maintaining a viable endothelium, which is crucial to successfultransplantation. Storage of the cornea for up to 14 days at 4° C. hasbeen reported, although the current technology does not permit adequatetissue preservation beyond a few days. Storage longer than 96 hours isattended by epithelial decomposition and loss of corneal clarity, asdemonstrated by increased swelling of the corneal stroma. This stromaledema is attributed to the decreased maintenance of the barrier pumpfunction of the corneal endothelium, a specific cell layer lining thecorneal stroma.

The functional status of the endothelium and sustained cornealdeturgescence after corneal preservation are of great clinicalimportance, and contribute primarily to the success of the surgicaloutcome. The ability of the cornea to maintain a relatively dehydratedstate is essential to the maintenance of corneal transparency. Cornealdeturgescence is an energy-dependent phenomenon performed primarily bythe endothelial cells. In order for the cornea to remain viable, variousenzymatic reactions must occur to carry out energy-dependent functions,maintained by high levels of ATP.

The lower temperature of the 4° C. corneal storage method reduces themetabolic rate of the cornea, but the storage medium must still be ableto support the basal requirements of the cornea. Thus, corneal storagemedia are a complex mixture of balanced salts, amino acids, energysources, antioxidants, buffering agents, cell membrane stabilizers,glycosaminoglycans, deturgescents and antibiotics. Temperature reductionchanges the membrane lipids, proteins and water structures, each ofwhich could alter the active transport mechanism by hindering the easeof passive diffusion, carrier-substrate interaction and energy-couplingrelationships. Thus disturbances of membrane function, as well asmorphological and biochemical alterations, assume a greater consequenceas the direct result of the lower metabolic rate. Therefore, a criticalevaluation of physiologic parameters such as ionic and amino acidcomposition, bicarbonate equilibrium, available energy sources,dissolved oxygen levels, osmolality and pH should be observed withrespect to each preservation medium. Parameters for extended 4° C.storage should be defined as to the reversibility of cell damageincurred during storage.

Adult corneal endothelium have a limited regenerative capacity andmitotic figures have been rarely observed in vivo; human cornealendothelium in vivo normally responds to trauma by sliding into thewounded area by cell migration. However, in vivo endothelial cellmitosis has been demonstrated in rabbits, cats and primates. In tissueculture, mitosis has been observed in rabbits and human cornealendothelium. Autoradiographic thymidine uptake studies aftercryowounding or mechanical wounding of corneas in vitro has demonstratedexistence of mitotic figures in the endothelial monolayer. Surgicaltrauma and disease can accelerate the loss of endothelial cells andfurther compromise the cornea. Thus, the long term preservation andenhancement of the corneal endothelium is a very important aspect of eyebank storage of eye tissue.

An overview of the issues surrounding the storage and handling ofcorneal tissue is found in Corneal Surgery, Chapters 1-4, pages 1-128edited by Federick S. Brightbill, M.D., published by C. V. MosbyCompany, St. Louis, Mo. 1986. A variety of storage media and techniqueshave been proposed, and current research continues to be directedtowards maintaining and actually enhancing the quality of donor tissues,and increasing the duration of storage corneal tissues, as defined asthe time between excision from a donor and transplantation.

Accordingly, the present invention is directed toward materials andmethods for enhancing ocular tissues, especially corneal tissues, duringstorage prior to transplantation. One aspect of the invention providesfor the enhancement of corneal tissue viability by maintaining normalphysiologic metabolism and corneal deturgescence during low temperaturestorage. Another aspect of the invention provides for increasing thelength of time that eye tissues, especially corneal tissues, canmaintain the attributes of fresh tissue.

SUMMARY OF THE INVENTION

Intermediate-term corneal storage at 4° C. should provide tissuepreservation which is capable of sustaining the functional status of theendothelium. Experimental work has demonstrated that both human andanimal eye tissues, especially corneas, are protected from deteriorationand actually are enhanced during low temperature eye bank storage in adefined serum-free, nutrient supplemented preservation solution. Theundesirable attributes of storage in serum-containing solutions areavoided, and the potential of the corneal endothelial cells to maintainnormal physiologic metabolism and corneal deturgescence during lowtemperature storage is increased.

The corneal endothelium is responsible for preservation of thetransparency of all corneal layers. The endothelium regulates the ioncomposition of the various corneal layers, thereby maintaining osmoticpressure, permitting permanent hydration of the cornea, and thusconstant thickness and transparency. Consequently, any disturbance ofendothelial cell function provokes corneal edema followed by partial orcomplete loss of transparency. The composition of synthetic media mustaddress the increased stromal hydration that occurs with increasedpreservation time and reduced temperatures.

The remarkable capacity of the corneal stroma to uptake water is due tothe presence of glycosaminoglycans (GAGS), such as chondroitin sulfate,dermatan or keratan sulfate between the collagen fibers. Electronmicroscopic studies comparing the collagen fibrils in swollen cornealstromas demonstrated that the diameter of collagen fibrils did notdiffer significantly from that of the normal fibrils. This findingsuggests that it is, rather, the volume increase of the interfibrillarsubstance which is responsible for the swelling of the stroma.Additional refraction studies demonstrated that the hydration of thefibrils is unchanged despite the the fact the cornea can swell from astate of near dryness to three times its normal thickness. When corneasare treated with hyaluronidase or cetylpyridinium chloride the stromalswelling is greatly reduced. These studies also suggest that theswelling takes place in the interfibrillar substances.

Glycosaminoglycans, such as chondroitin sulfate, are long, unbranchedpolysaccharide chains composed of repeating disaccharide units.Glycosaminoglycans are highly negatively-charged due to the presence ofsulfate or carboxyl groups, or both, on many of the sugar residues.Glycosaminoglycan chains tend to adopt highly extended, random coiledconformations, and to occupy a huge volume for their mass. Beinghydrophilic, they attract large amounts of water, thereby forminghydrated gels at even low concentrations. This tendency is markedlyenhanced by their high density of negative charges, which attractosmotically active cations. This water-attracting property ofglycosaminoglycans creates a swelling pressure, or turgor, in theextracellular matrix that resists compressive forces, in contrast tocollagen fibrils, which resist stretching forces. Because of theirporous and hydrated organization, the glycosaminoglycan chains allow therapid diffusion of water soluble molecules.

Recent studies suggest that not only has the proteoglycan groundsubstance as a whole been implicated as playing a significant role incorneal hydration, but that the specific distribution of the differentproteoglycans, which have different hydrating power, may also play arole in the establishment of water gradients across the cornea. Thedistribution of keratan sulfate and chondroitin-4-sulfate across thecornea directly relates to the asymmetric hydration of the cornea. Thereis a greater chondroitin-4-sulfate concentration near the epitheliumthan near the endothelium; keratan sulfate is more concentrated near theendothelium. Keratan sulfate and predominantly keratan sulfate-bearingproteoglycans have great water sorptive capacity, but meager waterretentive capacity. It is therefore plausible that the keratansulfate-bearing proteoglycan gradient, highest at the endothelium, helpsto set up the total water content gradient because of its great sorptivecapacity. In contrast, the chondroitin-4-sulfate and dermatansulfate-bearing proteoglycans, with their great water retentivecapacity, can help establish the bound water gradient that is maximumnear the epithelium. This gradient would then serve to diminish thedehydration of the front of the cornea, which is exposed to theatmosphere. Therefore, the water gradient across the cornea is highlycorrelative with the distribution of proteoglycans and their watersorptive and retentive capacities.

The present invention reduces intraoperative and postoperative reboundswelling associated with the use of chondroitin sulfate-containingpreservation solutions. The increase of corneal swelling may be due tothe influx of low molecular weight moieties of chondroitin sulfate intothe stroma during prolonged low temperature storage. Additional fluid isimbibed through the cut edge of the scleral-corneal rim. The use ofdeturgescent agents, such as dextran and increased concentrations ofchondroitin sulfate, control corneal hydration during low temperaturestorage. Dextran, a neutrally-charged molecule, osmotically restrictsexcess water from swelling the cornea during storage while chondroitinsulfate, a negatively charged molecule, actually binds to the cellmembrane and provides a membrane stabilizing effect. Chondroitin sulfateand dextran assist in the prevention of stromal hydration by increasingthe colloidal osmotic pressure in the aqueous environment surroundingthe stored cornea. Sustained corneal deturgescence during and aftercorneal preservation are of great clinical importance, reducing handlingand suturing problems encountered by the transplant surgeon, andconsequently reducing the risk of graft failure.

The functional status of the endothelium and sustained cornealdeturgescence after corneal preservation are of great clinicalimportance, and contribute primarily to the success of the surgicaloutcome. Other areas addressed in the present invention include theenhancement of corneal wound healing, and the reduction or eliminationof the normal progressive loss of endothelial cells, through the use ofnutritive cell supplements. Timely and adequate healing of cornealtissues is required to restore visual acuity.

There is a loss of corneal endothelial cells throughout life. Inaddition, endothelial cells are frequently damaged or destroyed inoperations involving the anterior chamber. Damage by trauma or lossthrough aging is compensated by growth in size of the endothelial cells,which migrate to cover denuded surfaces of Descemet's membrane. Inclinical cases, endothelial dysfunction is associated with variations ofcell size rather than cell density. The appearance of increased numbersof giant cells contributes greatly to increased corneal edema. Thejunctions of giant cells are abnormal. These abnormalities in celljunctions increase the permeability of the intercellular spaces, thusincreasing the fluid diffusion toward the cornea. The decreased densityof organelles, such as mitochondria or rough endoplasmic reticulum, arediminished in giant cells. These organelles are essential for theadequate functions of the biological pump. Insufficient pump functionresults in excess accumulation of fluid in the corneal stroma.Furthermore, these giant cells have extended external membranes,supporting functional changes associated with decreased biological pumpsites, associated with increased corneal swelling. It should be notedthat disturbances in endothelial cell function leading to corneal edemaoccur when endothelial cell density falls to 40% of the normal value,when hexagonality falls to 33%, when the coefficient of variation ofendothelial cell density increases three-to-four fold, and the size ofgiant cells has increased by 7.5 times over normal endothelial cells.

It is evident from these studies that the anterior chamber environmentlimits cell regeneration of the endothelium, and supports wound healingvia cell migration. Extreme cell loss is compensated by the formation ofgiant cells. Furthermore, it is the complex interaction of the humancorneal endothelial cell and the extracellular matrix that signal thecell to respond to cell loss in this manner.

The present invention further defines a nutritive solution that providesthe cornea with additional amino acids, vitamins, trace minerals, andenergy promoting precursors to enhance cell metabolism, wound healingand viability. Cell proliferation is regulated by events leading to DNAsynthesis; whether or not a cell proceeds with DNA synthesis or isarrested in the early stages of the cell cycle is dependent uponextracellular conditions. Cellular metabolism can be enhanced by theaddition of essential nutritive components by increasing hexosetransport, glycogen transport, protein synthesis, amino acid and iontransport.

The novel defined nutrient containing solutions are serumfree. Whileserum-supplemented solution can stimulate limited mitosis in humancorneal endothelial cells in tissue culture, the presence of serum inproducts for use with tissues for human transplantation presents manydisadvantages. Serum can be an agent for the transmission of diseases,such as viral diseases. Non-human-derived sera contains many substancescapable of eliciting response, and all sera contain some substances suchas endotoxins, and growth factors, that actually retard cell mitosis.These disadvantages are avoided by the present, serum-free solution.

Cornea preservation solutions are well known. In general, those employedherein contain an aqueous nutrient and electrolyte solution, aglycosaminoglycan, a deturgescent agent(s), an energy source(s), abuffer system(s), an antioxidant(s), membrane stabilizing components,antibiotic(s), ATP precursors and nutrient cell supplements. Nutrientand electrolyte solutions are well defined in the art oftissue-culturing. Such solutions contain the essential nutrients andelectrolytes at minimal concentrations necessary for cell maintenanceand cell growth. The actual composition of the solutions may varygreatly. In general, they contain inorganic salts, such as calcium,magnesium, iron, sodium and potassium salts of carbonates, nitrates,phosphates, chloride and the like, essential and non-essential aminoacids, vitamins and other essential nutrients.

Chemically defined basal nutrient media are commercially available, forexample from Gibco Laboratories (3175 Stanley Road, Grand Island, N.Y.14073) and Microbiological Associates (P.O. Box 127, Briggs Ford Road,Walkersville, Md. 21793) under the names Eagle's Minimal EssentialMedium (MEM) and TC199. Corneal storage solutions have been adapted fromthese nutrient media. The defined serumfree medical solution base of thepresent invention is composed of components found in both MEM and TC199supplemented with ATP precursors, vitamins, amino acids and growthpromoting supplements. The defined serumfree medical solution iscompared with commercially available corneal storage medium CSM™developed by R. L. Lindstrom, M.D. and Debra L. Skelnik, B.S., availablefrom Chiron Ophthalmics, Inc. (Irvine, Calif.) and TC199 from GibcoLaboratories (Grand Island, N.Y.) in Table I.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Preferred defined serumfree medical solutions for use in the compositionand methods of this invention contain an aqueous electrolyte solution(e.g. Minimal Essential Medium and/or TC199), a glycosaminoglycan (e.g.standard or purified high or low molecular weight chondroitin sulfate(A, B or C isomers), dermatan sulfate, dermatin sulfate, heparinsulfate, heparan sulfate, keratin sulfate, keratan sulfate and/orhyaluronic acid in a range of 0.01 mg/ml to 100 mg/ml; a deturgescentagent (e.g. low or high molecular weight polysaccharide, such asdextran, dextran sulfate, polyvinyl pyrrolidone, polyethylene glycol,polyvinyl acetate, hydroxypropylmethyl cellulose, carboxypropylmethylcellulose) in a range of 0.01 mg/ml to 100 mg/ml; an energy source andcarbon source (e.g. glucose, pyruvate, sucrose, fructose, dextrose) in arange of 0.05 mM to 10 mM; a buffer system (e.g. a bicarbonate buffersystem and hydroxyethylpiperizene ethanesulfonic acid, HEPES buffer) ina range of 0.1 mM to 100 mM; to maintain a physiologic pH (desirablybetween 6.8 and 7.6), an antioxidant (e.g. ascorbic acid,2-mercaptoethanol, glutathione, alpha tocopherol), in a range of 0.001mM to 10 mM; membrane stabilizing agents (e.g. vitamins A and B,retinoic and/or cofactors, ethanolamine, and phosphoethanolamine,selenium and transferrin), in a range of 0.01 mg/ml to 500 mg/ml;antibiotics-and/or antimycotic agents. (e.g. amphotericin-B, gentamycinsulfate, kanamycin sulfate, neomycin sulfate, nystatin, penicillin,tobramycin, streptomycin sulfate) in a range of 0.001 mM to 10 mM; andATP precursors (e.g. adenosine, inosine, adenine) in a range of 0.001 mMto 10 mM; and nutrient cell supplements (e.g. cholesterol,L-hydroxyproline, d-biotin, calciferol, niacin, para-aminobenzoic acid,pyridoxine HCl, Vitamin B12, Fe(NO₃)₃, non-essential amino acids) in arange of 0.001 mM to 10 mM.

The serumfree medical solution of this invention is composed of adefined aqueous nutrient and electrolyte solution, supplemented with aglycosaminoglycan(s), a deturgescent agent(s), an energy source(s), abuffer system(s), an antioxidant(s), membrane stabilizing components,antibiotic(s), ATP precursors and nutrient cell supplements in theamounts sufficient to enhance cell metabolism, cell viability, woundhealing, and corneal deturgescence following low temperature eye bankstorage. The excised corneas are aseptically transferred to containersof the corneal storage solution, which are then sealed. For storage andtransport,these corneas are maintained at low temperature (e.g. 2° C. to15° C. optimally at 4° C.) to minimize the risk of bacterial growth andto reduce corneal tissue metabolic damage. It has been found that evenat these low temperatures, the endothelial cells can be maintained forperiods up to 14 days. At the time of transplant-ation, normal cornealdeturgescence is maintained intraoperatively and postoperatively.Endothelial cell function and metabolism is maintained, permittingpermanent hydration of the cornea, and thus constant thickness andtransparency postoperatively. In addition to providing a viable corneafor transplantation, wound healing is protentiated. Variousmodifications can be made to the present invention without departingfrom the apparent scope thereof. For instance, the serumfree solutioncan be used in any medical application, and is not strictly limited toophthalmology. The invention is further illustrated by the followingexamples, which is not intended to be limiting.

BRIEF DESCRIPTION OF THE FIGURES

Table I: Formulation of TC-199, CSM™ and a representative formulation ofthe defined serumfree medical solution.

FIG. 1: Corneal Thickness of Human Corneas After 4° C. Storage

FIG. 2: Corneal Thickness After 12 Days Storage at 4° C. and PostStorage Warming to 24° C.

FIG. 3: [³ H]-Thymidine Incorporation of Human Corneal Endothelial CellsIncubated With Defined Serumfree Medical Solution Components

FIG. 4: Postoperative Corneal Thickness (mm)

MODE OF OPERATION Example One Defined Serumfree Medical Solution

Intermediate-term corneal storage at 4° C. should provide tissuepreservation capable of sustaining the functional status of theendothelium and the maintenance of corneal deturgescencepost-keratoplasty. CSM™ and K-Sol™ have become the standard media ofintermediate-storage at 4° C. As demonstrated in Kaufman H. E., VarnellE. D., Kaufman S. et al. K-Sol™ corneal preservation. Am J Ophthalmol1985. 100:299-304; Bourne W. M., Endothelial cell survival ontransplanted human corneas preserved at 4° C. in 2.5% chondroitinsulfate for one to 13 days. Am J Ophthalmol 1986; 102:382-6; LindstromR. L., Skelnik D. L., Mindrup E. A., et al: Corneal preservation at 4°C. with chondroitin sulfate containing medium. Invest Ophthalmol Vis Sci(Suppl) 1987; 28 (3): 167; Bhugra M. K., Sugar A., Meyer R., et al:Results of a paired trial of MK™ and K-Sol™ storage. Invest OphthalmolVis Sci (Suppl) 1988; 29: 112 and Lass J. H., Reinhart W. J., Bruner W.E., et al. Comparison of corneal storage in K-Sol™ and ChondroitinSulfate Corneal Storage Medium in human corneal transplantation.Ophthalmology 1989; 96: 688-97.

Increased corneal thickening is associated with CSM™-stored corneas,with greater rebound swelling apparent at the time of surgery. However,normal corneal thickness is achieved during the first post-operativemonth. The increase of corneal swelling may be due to the influx of lowmolecular weight moieties of chondroitin sulfate into the stroma duringprolonged storage at 4° C. In an effort to reduce corneal swelling,studies were conducted to determine if the addition of dextran to adefined serumfree chondroitin sulfate-containing medium would minimizecorneal hydration.

Dextran, an effective osmotic agent in MK™ medium, keeps the cornea thinand effectively maintains the barrier function of the cornealendothelium. Corneas stored in dextran-containing medium are inhibitedfrom swelling because of the colloidal osmotic pressure of dextran. Thedextran is present to osmotically restrict excess water from swellingthe cornea during storage. Dextran can penetrate the corneal endotheliumand enter the stroma. This entrance and egress of dextran occurs rapidlyat 4° C., with the degree of penetration of dextran depending on thelength of storage and the condition of the endothelium. Thus, dextranwas an attractive agent to reduce the corneal swelling associated withlow temperature storage with chondroitin sulfate containing medium.

The defined serumfree medical solution consisted of Eagle's MinimalEssential Medium (MEM) supplemented with Earle's salts, sodiumbicarbonate, 25 mM HEPES, 0.1 mM non-essential amino acids, 1 mM sodiumpyruvate, 2 mM L-glutamine, 0.5 mM 2-mercaptoethanol, 1.0% dextran, 2.5%chondroitin sulfate and 100 μg/ml gentamycin sulfate. The base mediumwas further supplemented with the following components: Fe(NO₃)₃ 9H₂ O,adenine sulfate, cholesterol, L-hydroxy tocopherol phosphate, D-biotin,calciferol, niacin, paraminobenzoic acid, pyridoxine HCl, adenosine,inosine, and vitamin B12. These components were added to more completelydefine the basal medium and potentiate cell growth and cell function(See Table I).

In order to determine the safety and efficacy of this defined serumfreemedical solution, a dose response curve of chondroitin sulfateconcentration with human corneas stored for 12 days at 4° C. wasconducted. Chondroitin sulfate concentrations consisted of 1.5%, 1.75%,2.0% and 2.5%. Corneal thickness measurements were taken at 0, 1, 7 and12 days storage at 4° C.

In addition, isolation techniques developed in our laboratory haveenabled the establishment of primary and subsequent subcultures of humancorneal endothelium that retain the attributes of native endothelium. Invitro conditions maintain these human corneal endothelial cells in aproliferative state, actively undergoing mitosis. A quantitativebioassay has been developed to determine the effects of various testmedium in the stimulation or inhibition of DNA synthesis as measured by[³ H]-thymidine incorporation.

Next a prospective pilot clinical trial was conducted, evaluatingcorneal thickness and endothelial cell survival for corneas that hadbeen stored in a defined serumfree medical solution (Formula A) and thentransplanted into patients.

MATERIALS AND METHODS Chondroitin Sulfate Dose Response Curve With HumanCorneas

Human donor globes were immersed in 1.0% povidone iodine in normalsaline for three minutes, followed by a one-minute immersion in normalsaline. The globes were then rinsed with 12 cc of normal saline with asyringe fitted with a 18-gauge needle. Sixteen paired corneas fromdonors unsuitable for transplantation because of age or cause of deathwere removed at a certified eye bank an average of 12.0 hours afterdeath, and placed in 20 ml of defined medical solution supplemented with1.5%, 1.75%, 2.0%, and 2.5% chondroitin sulfate. Control media wascommercial Dexsol™ (Chiron Ophthalmics, Inc., Irvine, Calif.).Supplemented media was warmed to room temperature before the corneaswere placed into the media, and corneal thickness measurements weretaken. Corneal thickness measurements were made using a Leitz uprightmicroscope fitted with a micrometer. The micrometer dial indicator wasattached to the microscope stand above the stage, with the set screwplacement through the stage, directly under the foot of the dialindicator. The corneal thickness measurement involved focusing on theendothelium, setting the set screw to bring the dial to `zero`, raisingthe stage to bring the epithelium into focus, and recording the dialindicator reading. The cornea was then cooled to 4° C., and stored for12 days. Corneas were removed from the storage medium and placed in 15ml of MEM supplemented with 2 mM L-glutamine and 100 μg/ml gentamycin.Corneas were then warmed to 34° C. for 2 hours and central cornealthickness measurements were taken at 30, 60 and 120 post-warming.Corneal endothelium was evaluated by staining with 0.1% trypan blue andalizarin red S after final corneal thickness measurements were taken.

[³ H]-Thymidine Incorporation of Human Corneal Endothelial Cells

Fourteen medium components were tested as follows: Fe(NO₃)₃ 9H₂ O,adenine sulfate, cholesterol, L-hydroxyproline, ascorbic acid, alphatocopherol phosphate, D-biotin, calciferol, niacin, para-aminobenzoicacid, pyridoxine HCl, adenosine, inosine, and vitamin B12. Componentswere added individually or in combination to a base medium consisting ofEagle's Minimal Essential Medium (MEM) supplemented with Earle's salts,25 mM HEPES, sodium bicarbonate, 0.1 mM non-essential amino acids, 1 mMsodium pyruvate, 2 mM L-glutamine, 0.5 mM 2-mercaptoethanol, 2.5%chondroitin sulfate and 100 μg/ml gentamycin sulfate. Additionalchondroitin sulfate concentrations of 1.75% and 2.0% were also tested.Control media consisted of commercially available Dexsol™ (ChironOphthalmics, Inc., Irvine, Calif.) and CSM™ supplemented with 10% fetalbovine serum. All test media samples were freshly made up and warmed toroom temperature at the time of the experiment.

QUANTITATIVE BIOSSAY

The quantitative bioassay is based on the incorporation of [³H]-thymidine into the DNA of human corneal endothelial cells incubatedin serumfree and serum containing medium. Costar 96-well tissue cultureplates were seeded with 3×10³ in a final volume of 200 μl of designatedmedium. Fourth passage human corneal endothelial cells were maintainedin a humidified incubator at 35.5° C. in a 95% air: 5% CO₂ atmosphere.After 24 hours of incubation in CSM™, supplemented with 10% fetal bovineserum, to permit attachment, the medium was removed, and each well wasrinsed once with serumfree Minimal Essential Medium with Earle's saltsand 25 mM HEPES. The cells were then rinsed and incubated with theappropriate test solution. Human corneal endothelial cells were thenIncubated for an additional 72 hours in the presence of 1microcurie/well of [³ H]-thymidine. Uptake was ended by the aspirationof the radioactive medium and rinsing the cells twice with serumfreeMinimal Essential Medium. The human corneal cells were detached with0.5% trypsin and prepared for liquid scintillation counting. The [³H]-thymidine counts represent acid-insoluble counts. One-way analysis ofvariance and the Newman-Keuls multiple range test were used to evaluatestatistical significance (p<0.05).

CLINICAL TRIAL

Eye Bank Procedures

Human donor globes were immersed in 1.0% povidone iodine in normalsaline for three minutes, followed by a one-minute immersion in normalsaline. The globes were then rinsed with 12 cc of normal saline with asyringe fitted with a 18-gauge needle. Corneas from suitable donors wereremoved at tile eye bank an average of 8.6 hours after death, and placedin a defined serumfree medical solution (Formula A). This solution waswarmed to room temperature before the cornea was placed into thesolution. The cornea was then cooled to 4° C., and stored for an averageof 4.3 days (range 1-7 days).

Recipient Criteria

The following recipient diagnoses were considered for entry into thestudy: aphakic bullous keratopathy, Fuchs' dystrophy, pseudophakicbullous keratopathy, corneal scar, keratoconus and failed graft. Thepreoperative examination consisted of measurement of best correctedvisual acuity, intraocular pressure, slit lamp and funduscopicexamination. Informed consent was obtained from all participants inclinical trials consistent with the United States Department of Healthand Human Services guidelines. This randomized clinical trial wasperformed with Institutional Review Board consent and monitoring.

Surgical Technique

Corneas were warmed to room temperature at the time of transplantation.The donor buttons were cut from the endothelial side with a cornealtrephine press. Sodium hyaluronate (Healon) or sodium hyaluronate withchondroitin sulfate (Viscoat) was used in all cases. Operative andpostoperative care was similar for all cases. Suturing techniquesconsisted of a combination of 12 interrupted 10-0 sutures with a running11-0 nylon or mersilene suture. Gentamycin, Betamethasone and Ancef wereinjected subconjunctivally at the end of each procedure.

Postoperative Treatment

Postoperatively all patients received neomycin or gentamycin drops fourtimes daily during the first month. Topical steroids were administeredas needed. Patients were evaluated during the first two monthspostoperatively for complications, rejection, corneal vascularization,infection, wound leak, dehiscence of wound, persistent epithelialdefects, and overall corneal condition. Ultrasonic pachymetry of thecentral cornea was performed preoperatively and postoperatively at oneday, one week, one month and two months. The total number of patientsincluded in this study was 15. Between group differences in cornealthickness were analyzed to determine if there were significantdifferences using a paired t-test.

RESULTS AND DISCUSSION

Dextran Dose Response Curve With Human Corneas

The chondroitin sulfate dose response curve for human corneas incubatedat 4° C. for 12 days with respect to corneal thickness is shown inFIG. 1. Corneas incubated with Dexsol™, containing 1.35% chondroitinsulfate, demonstrated effective thinning at 1, 7, and 12 days. Cornealthickness measurements at these time periods were 0.425±0.082 mm,0.530±0.040 mm and 0.572±0.043 mm. Corneas incubated with 1.5%-2.0%chondroitin sulfate demonstrated increased corneal deturgescence atthese same time periods with the greatest corneal thinning occurring at2.5% chondroitin sulfate. Corneal thickness at 1, 7, and 12 dayspost-incubation was 0.405±0.021 mm, 0.480±0.042 mm, 0.480±0.028 mm,respectively. Corneas stored in Dexsol™ for 12 days exhibited a 19.6%increase in corneal swelling post warming to 34° C. Corneas stored in1.5%-1.75% chondroitin sulfate demonstrated a statistically similarincrease in corneal swelling post-warming. Corneas stored in 2.0% and2.5% chondroitin sulfate demonstrated a 15.6% and 13.5% increase incorneal swelling post-warming (FIG. 2).

All endothelial cell monolayers were intact, with normal endothelialcell morphology for all concentrations of chondroitin sulfate tested.Corneas incubated with higher concentrations of chondroitin sulfatedemonstrated fewer stromal folds, and fewer areas of alizarin red Sstaining of Descemet's membrane. All alizarin red S staining was minimalfor all corneas, and was confined to areas of stromal folding. Inconclusion, all corneas stored in 1.35%-2.5% chondroitin sulfate hadintact corneal endothelium after 12 days preservation at 4° C., Corneasstored in the defined medical solution (containing 2.5% chondroitinsulfate) maintained the greatest corneal deturgescence over the 12 daypreservation period, Minimal corneal folding and swelling was also notedfor this test group after rewarming to 34° C. These results support theuse of this defined serumfree medical solution to preserve human corneasat 4° C. for transplantation.

[³ H]-Thymidine Incorporation of Human Corneal Endothelial Cells

This study was conducted to evaluate the components of a definedserumfree medical solution. The test medium was evaluated in a [³H]-thymidine incorporation bioassay with human corneal endothelialcells. This bioassay provides a sensitive method to determine if thetest medium will inhibit or stimulate the incorporation of [³H]-thymidine into the DNA of these cells. The incorporation of [³H]-thymidine by human corneal endothelial cells incubated with testmedium containing one or more of fourteen components was compared toserumfree Dexsol™ medium and CSM™ medium supplemented with 10% FBS (FIG.3). One-way analysis of variance and the Newman-Keuls multiple rangetest were used to evaluate statistical significance (p<0.05).

In this bioassay, the cells were kept in a proliferative state, activelyundergoing mitosis. Inhibition of [³ H]-thymidine incorporation into theDNA of human corneal endothelial cells is an indicator of decreased cellmetabolism, decreased cell health and possible cellular toxicity. Humancorneal endothelial cells incubated with CSM™ medium supplemented with10% FBS exhibited a statistically significant Increase in [³H]-thymidine incorporation rate as compared to the freshly preparedcontrol serumfree Dexsol™ medium. HCE cells incubated with 1.75% or 2.0%chondroitin sulfate exhibited statistically similar [³ H]-thymidineincorporation rates as HCE cells incubated with serumfree Dexsol™. Theaddition of 2.5% chondroitin sulfate and 1% dextran, in combination withthe following individual components: Fe(NO3)3.9H20, adenine sulfate,L-hydroxyproline, ascorbic acid, alpha tocopherol phosphate, D-biotin,pyridoxine HCl, inosine, and vitamin B12 exhibited statistically similarrates of [³ H]-thymidine incorporation as HCE cells incubated withserumfree Dexsol™. The addition of 2.5% chondroitin sulfate and 1%dextran, with adenosine or combination of adenosine, adenine, andinosine exhibited statistically greater [³ H]-thymidine incorporationrates than HCE cells incubated with the Dexsol™ control medium. When allfourteen components were combined with 2.5% chondroitin sulfate in asupplemented MEM base, a statistically greater [³ H]-thymidineincorporation rate was demonstrated as compared to the Dexsol™ control.All media tested maintained normal endothelial cell morphologythroughout the 72-hour incubation period.

In conclusion, from the results of this [3H]-thymidine incorporationstudy with human corneal endothelial cells, a defined serumfree solution(Formula A) containing: 2.5% chondroitin sulfate, 1% dextran,Fe(NO3)3.9H20, adenine sulfate, cholesterol, L-hydroxyproline, ascorbicacid, alpha tocopherol phosphate, D-biotin, calciferol, niacin,para-aminobenzoic acid, pyridoxine HCl, adenosine, inosine, and vitaminB12 was capable of stimulating [³ H]-thymidine incorporation ratesstatistically greater than serumfree Dexsol™ medium as defined by theparameters of this bioassay. This defined serumfree medical solution iscapable of enhancing the mitotic potential of human corneal endothelialcells, by providing a more completely defined solution than the controlDexsol™ medium. This solution is therefore, acceptable for use as a 4°C. corneal preservation medium.

Clinical Study

Fifteen corneas were transplanted utilizing the defined serumfreemedical solution (Formula A). All patients were operated on by onesurgeon and were included in the following study. The cornea donors hadthe following characteristics: donor age (mean age 53±19 years), deathto enucleation time (mean: 4.3±2.7 hours), and death to preservationtime (mean: 4.3±3.2 hours). Storage time of corneas at 4° C. was 4.3days (range 1-7 days). One-hundred percent of the Formula A transplantedcorneas were clear after 2 months. No persistent epithelial defects werenoted in this patient group. Intraoperative corneal thickness was0.623±0.054 mm. Comparative corneal intraoperative thicknessmeasurements of corneas stored in Dexsol™ under similar parameters was0.787±0.047 mm. Corneal thickness measurements at one week for Formula Aand Dexsol stored corneas was 0.650±0.084 mm and 0.743±0.093 mm,respectively. Formula A stored corneas were significantly thinnerintraoperatively and at one week post-operatively (FIG. 4). Progressivecorneal thinning occurred for all patients during the 2 month follow-upperiod (corneal thickness: one month 0.612±0.167 mm; two months0.544±0.062 mm). Post-operative intraocular pressures were within normallimits for all patients. No primary donor failures occurred in thisFormula A cornea group.

The defined serumfree medical (Formula A) solution was effective inmaintaining normal corneal deturgescence intraoperatively andpost-operatively. Endothelial cell function and metabolism wasmaintained, permitting normal hydration of the cornea, and thussustaining constant corneal thickness and transparency post-operatively.

    TABLE I       TC199 CSM Formula A  M-K CSM Formula A  M-K CSM Formula A Medium mg/L     mg/L mg/L  mg/L mg/L mg/L  mg/L mg/L mg/L       INORGANIC SALTS    AMINO ACIDS    VITAMINS    CaCl2 (anhyd) 140 200     200 DL Alanine 50   Ascorbic acid 0.05  0.05 Fe(NO3)3 9H2O 0.72  0.5 L     Alanine  8.9 8.9 Alpha Tocopherol Phosphate 0.01  0.01 KCl 400 400 400 L     Arginine HCl 70 126 126 (Disodium salt) KH2PO4 60   L Asparagine H2O  15     15 d-Biotin 0.01  0.01 MgSO4 7H2O 200 200 200 DL Aspartic acid 60     Calciferol 0.1  0.1 NaCl 8000 6800 6800 L Aspartic Acid  13.3 13.3 D-Ca     pantothenate 0.01 1 1 NaHCO3  2200 2200 L Cysteine HCl H2O 0.11     Choline Chloride 0.5 1 1 NaH2PO4 H2O  140 140 L Cystine 26 24 24 Folic     acid 0.01 1 1 Na2HPO4 7H2O 90   DL Glutamic acid H2O 150   I-Inositol     0.05 2 2     L Glutamic acid  14.7 14.7 Menadione 0.01 OTHER COMPONENTS       L Glutamine 100 292 292 Niacin 0.025  0.025 Adenine Sulfate 10  10     Glycine 50 7.5 7.5 Niacinamide 0.025 Adenosinetriphosphate-    L     Histidine HCl H2O 21.88 42 42 Nicotinamide  1 1 (Disodium Salt) 1   L     Hydroxyproline 10  10 Para-aminobenzoic acid 0.05  0.05 Adenylic Acid     0.2   DL Isoleucine 40   Pyridoxal HCl 0.025 1 1 Cholesterol 0.2  0.2 L     Isoleucine  52 52 Pyridoxine HCl 0.025  0.25 Deoxyribose 0.5   DL     Leucine 120   Riboflavin 0.01 0.1 0.1 D-Glucose 1000 1000 1000 L Leucine      52 52 Thiamine HCl 0.01 1 1 Glutathione (reduced) 0.05   L Lysine HCl     70 72.5 72.5 Vitamin A (acetate) 0.014 Guanine HCl 0.3   DL Methionine     30 Hypoxanthine 0.3   L Methionine  15 15 Adenosine   5 (or) Hypoxanthine     -Na salt 0.344   DL Phenylalanine 50   Inosine   10 Phenol Red 20 10 10     L Phenylalanine  32 32 Vitamin B12   1.36 Ribose 0.5   L Proline 40 11.5     11.5 Sodium acetate 50   DL Serine 50   Additional Components Sodium     Pyruvate  110 110 L Serine  10.5 10.5 2-mercaptoethanol  .5 mM .5 mM     Thymine 0.3   DL Threonine 60   Chondroitin Sulfate %  1.35 2.5 Tween 80     20   L Threonine  48 48 Gentamycin μg/ml  100 100 Uracil 0.3   DL     Tryptophan 20   Dextran %   1 Xanthine 0.3   L Tryptophan  10 10     L     Tyrosine 40 36 36     DL Valine 50     L Valine  46 46

We claim:
 1. The defined serumfree medical solution consistingessentially of effective amounts of:a. an aqueous nutrient andelectrolyte solution; b. a glycosaminoglycan; c. a deturgescent agent;d. an energy source; e. a buffer system; f. an antioxidant; g. membranestabilizing agents; h. an antibiotic and/or an antimycotic agent; i. ATPpresursors; and j. nutrient cell supplements having improved cornealpreservation upon storage as compared to the same composition notcontaining the (g) or (h) or (i) or (j) components.
 2. The definedserumfree medical solution containing components which maintain andenhance the preservation of eye tissues, including human corneal tissuesat low temperatures (2° C. to 15° C.) with a physiological pH consistingessentially of effective amounts of:a. an aqueous nutrient andelectrolyte solution; b. a glycosaminoglycan; c. a deturgescent agent;d. an energy source; e. a buffer system; f. an antioxidant; g. membranestabilizing agents; h. an antibiotic and/or an antimycotic agent; i. ATPpresursors; and j. nutrient cell supplements having improved cornealpreservation upon storage as compared to the same composition notcontaining the (g) or (h) or (i) or (j) components.
 3. The definedserumfree medical solution containing components which maintain andenhance the preservation of eye tissues, Including human corneal tissuesat low temperatures (2° C. to 15° C.) with a physiological pH comprisedof:a. An aqueous nutrient and electrolyte solution selected from thegroup of:1. Eagle's minimal essential medium (MEM)
 2. TC199 medium
 3. Acombination of Eagle's minimal essential medium (MEM) and TC199 b. Aglycosaminoglycan in the range of 0.01 mg/ml to 100 mg/ml selected fromthe group of:1. chondroitin sulfate;
 2. dermatan sulfate;
 3. dermatinsulfate;
 4. heparin sulfate;
 5. heparan sulfate;
 6. karatin sulfate; 7.keratan sulfate; and/or
 8. hyaluronic acid; c. A deturgescent agent inthe range of 0.01 mg/ml to 100 mg/ml selected from the group of:1.dextran;
 2. dextran sulfate;
 3. polyvinyl pyrrolidone;4. polyethyleneglycol;
 5. polyvinyl acetate;
 6. hydroxypropylmethyl cellulose; and 7.carboxypropylmethyl cellulose; d. An energy source in a range of 0.05 mMto 10 mM selected from the group of:1. glucose;
 2. pyruvate;
 3. sucrose;4. fructose; and
 5. dextrose; e. A buffer system in a range of 0.1 mM to100 mM selected from the group of:1. Bicarbonate buffer; and
 2. HEPESbuffer; f. An antioxidant in a range of 0.001 mM to 10 mM selected fromthe group of:1. ascorbic acid;
 2. 2-mercaptoethanol;
 3. glutathione; and4. alpha-tocopherol; g. A membrane stabilizing component in a range of0.01 mg/ml to 500 mg/ml selected from the group of:1. vitamin A; 2.vitamin B;3. retinoic acid;
 4. ethanolamine;
 5. phosphoethanolamine; 6.selenium and
 7. transferrin; h. An antibiotic and/or antimycotic in therange of 0.1μg/ml to 1 mg/ml selected from the group of:1.amphotericin-B;
 2. gentamycin sulfate;
 3. kanamycin sulfate;
 4. neomycinsulfate;
 5. nystatin;
 6. penicillin;
 7. tobramycin; and
 8. streptomycin;i. ATP presursors in a range of 0.001 mM to 10 mM selected from thegroup of:1. adenosine;
 2. inosine; and
 3. adenine; j. Nutrient cellsupplements in a range of 0.001 mM to 10 mM selected from the groupof:1. cholestrol;
 2. L-hydroxyproline;
 3. d-biotin;4. calciferol; 5.niacin;
 6. para-aminobenzoic acid;
 7. pyridoxine HCl;
 8. vitamin B12; 9.Fe(NO₃)₃ ; and
 10. non-essential amino acids having improved cornealpreservation upon storage as compared to the same composition containingthe (g) or (h) or (i) or (j) components.
 4. The defined serumfreemedical solution containing components which maintain and enhance thepreservation of eye tissues, including human corneal tissues at lowtemperatures (2° C. to 15° C.) with a physiological pH consistingessentially of effective amounts of:a. An aqueous nutrient andelectrolyte solution:1. Eagle's minimal essential medium (MEM) b. Aglycosaminoglycan in the range of 0.01 mg/ml to 100 mg/ml1. chondroitinsulfate; c. A deturgescent agent in the range of 0.01 mg/ml to 100mg/ml1. dextran; d. An energy source in a range of 0.05 mM to 10 mM1.pyruvate;
 2. dextrose; e. A buffer system in a range of 0.1 mM to 100mM1. Bicarbonate buffer; and
 2. HEPES buffer; f. An antioxidant in arange of 0.001 mM to 10 mM1. 2-mercaptoethanol; and
 2. alpha-tocopherol;g. An antibiotic and/or antimycotic in the range of 0.1 μg/ml to 1mg/ml1. gentamycin sulfate; h. ATP presursors in a range of 0.001 mM to10 mM
 1. adenosine;2. inosine; and
 3. adenine; i. Nutrient cellsupplements in a range of 0.001 mM to 10 mM1. cholestrol; 2.L-hydroxyproline;
 3. d-biotin;
 4. calciferol;
 5. niacin; 6.para-aminobenzoic acid;
 7. pyridoxine HCl;
 8. vitamin B12;
 9. Fe(NO₃)₃ ;and
 10. non-essential amine acids.
 5. A method for the storage andpreservation of corneal tissue, the method comprising bringing thetissue into contact with the solution of claim 1.